Microorganisms, and their enzymes, have long been utilized as biocatalysts in the preparation of various products. The action of yeast in the fermentation of sugar to ethanol is an immediately recognizable example. In recent years, there has been a growing interest in the use of microorganisms and their enzymes in commercial activities not normally recognized as being amenable to enzyme use. One example is the use of microorganisms in industrial processes, particularly in the treatment of waste products.
Nitrile-containing compounds are used in a wide variety of commercial applications. For example, nitriles are used in the synthesis of many commercially useful compounds including amines, amides, amidines, carboxylic acids, esters, aldehydes, ketones, imines, and heterocyclics. Nitriles also are used as solvents, as herbicides, and in the synthesis of detergents and antiseptics. One of the more commercially important nitriles is acrylonitrile, which is used in the production of acrylamide, acrylic acid, acrylic fibers, copolymer resins, and nitrile rubbers.
The waste streams generated in the production of nitriles often contain high concentrations of hazardous nitrogen-containing compounds. For example, the waste streams can contain nitriles, such as acetonitrile, acrylonitrile, succinonitrile, and fumaronitrile. Further, such waste streams may also contain hazardous compounds, such as cyanides, acrylamides, acrolein, and cyanohydrins. As hazardous wastes generally cannot be released legally into the environment, methods for treating waste streams to remove or remediate one or more hazardous components are important in commercial production processes.
One method for treating nitrogen waste streams has been through the use of certain microorganisms that convert nitrile compounds into their corresponding amides or acids. For example, U.S. Pat. Nos. 3,940,316 and 4,001,081 disclose the use of nitrile hydratase microorganisms to produce acrylamide from acrylonitrile.
Generally, nitrile converting microorganisms degrade aliphatic nitriles in a two step reaction involving nitrile hydratase and amidase. In a first step, nitrile hydratase catalyzes the hydrolysis of the nitrile (or cyanohydrin) to the corresponding amide (or hydroxy acid). In a second step, amidase catalyzes the hydrolysis of the amide to the corresponding acid or hydroxy acid. Similarly, some microorganisms have been shown to degrade aromatic nitriles by directly converting these nitriles to their respective acid through the action of nitrilase.
Since the initial reports documenting the potential commercial utility of the biological conversion of acrylonitrile to acrylamide, the enzymes involved in the microbial degradation of nitriles have received considerable interest. The possibility of enzymatic preparation of chiral acids (such as hydroxy acids from cyanohydrin precursors) has also been a focus of much interest in this field. Despite promising results, the various potential applications of the nitrile hydratase/amidase conversion discussed above have not yet been fully exploited.
Another example of the growing use of microorganisms and their enzymes is in the formation of aspartic acid. Asparaginase I is an enzyme that catalyzes the hydrolysis of asparagine to aspartic acid, as shown below:HOOCCHNH2CONH2+H2O→HOOCCHNH2CH2COOH+NH3 
Asparaginase I can be found in bacteria, plants, and many animals; however, as human white blood cells do not possess the necessary asparagine synthase enzyme, the cells cannot make asparagine. It has thus been found that asparaginase I can be effective in the treatment of human malignant leukemia. Leukemia cells typically have low levels of asparagine synthase, the enzyme sometimes being completely absent. Leukemia cells, therefore, generally require an external source of asparagine. Since asparaginase I converts asparagine to aspartic acid, administering asparaginase I to a patient suffering from leukemia further limits the available source of asparagine for the cancerous cells and functions to weaken the cell making them more susceptible to chemotherapeutic treatments. Accordingly, asparaginase I is typically administered to a leukemia patient as part of a combination therapy with a chemotherapeutic agent.
Asparaginase I for use in such treatment is presently obtained from E. coli bacteria (in the form of a heterotetramer) and Erwinia bacteria (in the form of a homotetramer), but these sources each have disadvantages. For example, the asparaginase I obtained from E. coli is less effective than the asparaginase I obtained from Erwinia. However, it is much more difficult to produce asparaginase I using Erwinia than with E. coli. Further, these sources can result in the presence of Gram-negative toxins in the isolated enzyme, which is undesirable. Thus, there remains a need to increase asparaginase I production from a variety of microorganisms while avoiding simultaneous production of gram negative toxins, which can be harmful.
Stability, which is a key element for a practical biological catalyst, has been a significant hurdle to using nitrile hydratase and/or amidase in many commercial applications. While immobilization and chemical stabilizing agents are recognized approaches for improving enzyme stability, the current immobilization and stabilization techniques are still in need of further development. Accordingly, there remains a need in the art for method of inducing higher levels of enzymatic activity in a variety of microorganisms, particularly microorganisms capable of producing enzymes useful in the degradation of nitrile-containing compounds. Further, there is also a need for a method to improve the stabilization of key enzymes in the degradation of nitrile-containing compounds.